plant surface microbiology.pdf

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Thomas F.C. Chin-A-Woeng et al.
free of contamination.After incubation for 24 h on agar-solidified plant nutrient
solution (PNS) medium at 4 °C, seeds are allowed to germinate at 28 °C.
Seedlings are inoculated 2 days later. A F.o.r.l. spore suspension, prepared as
described previously, is added to the plant nutrient solution to a final concentration
of 5x10 2 spores/ml, which is than mixed through the sterile sand to
10 % (v/w) PNS.
Rhizoctonia solani was grown on 2 % water agar for 5 days. Discs of approximately
4 mm in diameter were cut from the edge of a growing colony and
blended in PNS. P. ultimum was grown for 3–4 weeks in clarified V8 medium
or hemp seed extract in water for 1–2 weeks. Oospores were collected free of
the mycelium by washing them three times with sterile water and blending in
0.1 M sucrose. The blended culture was incubated for 2 h on a shaker
(130 rpm) at 28 °C, sedimented, and resuspended in 1 M sucrose. To kill the
mycelium fragments, the suspension was incubated at –20 °C for 12 h. The culture
was washed, layered over 1 M sucrose and centrifuged at 2300 rpm.
Oospores were added in a final concentration of 5–25 oospores/g of sand.
Germinated tomato seeds were incubated in a bacterial suspension with a
concentration of 10 7 CFU/ml (Pseudomonas) or 10 9 CFU/ml (Bacillus) for
10 min, after which the germinated seeds were planted in the sand at a depth
of approximately 5 mm. Seed inoculation is preferred above inoculation from
soil since commercial biocontrol of tomato pathogens is also based on seed
coating while the pathogen is already present in the soil. This form of inoculation
also results in more reproducible experimental data. Plants were grown
in a growth chamber or a greenhouse for 7 days and the disease index was
determined by scoring the plants according a fixed disease index (Table 1).
The data can be analysed statistically using a standard c 2 analysis.
To confirm the presence of the fungus on plants, suspected diseased root
parts can be placed in 0.005 % household bleach for 30 s, thoroughly rinsed
with sterile water, and placed on a rich (LC or PDA) medium. Plates are
inspected for fungal growth after incubation at 28 °C for 2 days.
Table 1. Example of Pythium ultimum disease indices
Disease symptoms Disease index
No visible symptoms 0
Small brown spots on the main root and/or the crown 1
Brown spots on the central root and extensive discoloration of crown 2
Damping-off 3
Dead 4